Apparatus, system, and method for electrochemical pump-based chromatography separations in microfabricated devices

ABSTRACT

Rapid microchip LC analysis with integrated electrolysis-based pumping is achieved with a monolithic microfluidic chip. A pressure-balanced sample injection approach allows introduction of pL-range sample volumes without valves or other components that are difficult to integrate in microdevices. The approach also eliminates dead volume between injection and separation. On-chip LC separation of amino acids with elution times of &lt;40 s and good efficiency (3350 theoretical plates) is provided.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation-in-part of and claims priority to U.S. Provisional Patent Application No. 60/963,794 entitled “Electrically actuated, pressure-driven liquid chromatography separations in microfabricated devices” and filed on Aug. 6, 2007 for Adam T. Woolley et al., which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to microfluidic devices and more particularly relates to monolithic pressure-driven chromatography Microsystems.

2. Description of the Related Art

Miniaturization of analytical techniques holds great potential for performing a variety of sample-limited assays, especially because of the possibility of integrating multiple analysis steps in a single substrate. Since first demonstrated in 1992, capillary electrophoresis in microfabricated devices has seen significant advances, and numerous chemical and biological applications have been reported. Electrically driven methods are well-suited for miniaturization, since electroosmotic flow (EOF) can be controlled without valves or external pumps. However, EOF pumping and fluid transport have inherent issues that may limit their broad application in miniaturized methods. For example, EOF typically requires high voltages (kV) and is affected by Joule heating. Moreover, EOF is sensitive to the solution pH and column surface charge. Finally, electrophoretic techniques are optimal for charged analytes that can be exposed to an electric field.

On the other hand, pressurized separation methods such as liquid chromatography (LC) are more general and broadly used. In 1990, Manz et al. presented advantages of the miniaturization of LC. Even though no experimental data were provided, this paper showed that theoretically, the performance per unit time should be superior in microchip compared to conventional LC. (See A. Manz, Y. Miyahara, J. Miura, Y. Watanabe, H. Miyagi and K. Sato, Sens. Actuators B, 1990, 1, 249.) Manz and others have indicated that there are three critical elements for a fully miniaturized LC system. They are: (i) integration of a pumping mechanism capable of generating pressure and flow compatible with microchannel dimensions; (ii) incorporation of a separation medium inside microchannels; and (iii) a minimal dead volume interface of the separation column with the pumping and injection mechanism. (See Manz et al.; C. M. Harris, Anal. Chem., 2003, 75, 64A; and A. de Mello, Lab Chip, 2002, 2, 48N.) However, the miniaturization of pressure-driven separation methods presents challenges the solution to which is not trivial. Most reports have focused on micromachining a separation column while maintaining an external pumping mechanism. Thus, the full advantages of LC miniaturization have not been realized prior to the discoveries disclosed herein.

Two recent studies have made important progress in the development of miniaturized LC systems with integrated pumping and injection. Lazar et al. fabricated a LC microdevice with integrated EOF micropumps for sample valving and separation. (See I. M. Lazar, P. Trisiripisal and H. A. Sarvaiya, Anal. Chem., 2006, 78, 5513.) However, this approach required the use of relatively high electric fields (500 V/cm), was limited to solutions with low organic solvent concentrations, and was relatively slow (˜40 min elution times). In other work, a hybrid silicon-parylene microfluidic chip with integrated electrochemical micropumps for sample injection and separation was used for the LC analysis of protein digests. This report demonstrated the advantages of having a minimal dead volume between injection and separation, but again suffered from long analysis times (˜1 h). (See J. Xie, Y. Miao, J. Shih, Y.-C. Tai and T. D. Lee, Anal. Chem., 2005,77,6947.)

A variety of micropumps have been constructed for microfluidic applications. Mechanical pumps use moving parts, have relatively complex fabrication and often face compatibility challenges with regard to solutions and samples when integrated with microfluidic systems. Non-mechanical pumps based on electroosmotic, magnetohydrodynamic or electrochemical actuation, are thus appealing alternatives. Electroosmotic pumps, which generate pressure with EOF, are perhaps the most widely used micropumps in microfluidics applications. However, to obtain appropriate flow rates, it is often necessary to apply high voltages (˜kV) and make either packed small-diameter columns or microchannel network arrays, which complicate the fabrication process. Moreover, electroosmotic pumps are only suitable for operation within a certain range of solution pH and conductance values.

Column technology for microchip LC is a key challenge. Packing microchannels with particles as in conventional LC is difficult to achieve on the micro level due to pressure constraints and difficulties in forming frits inside microchannels. In 1998, Regnier et al. demonstrated surface-modified micromachined posts, as a mimic of a packed microcolumn. (See B. He, N. Tait and F. Regnier, Anal. Chem., 1998, 70, 3790.) While this approach is compatible with micromachining techniques, it is hindered by expensive fabrication protocols involving deep reactive ion etching. The possibility of performing separations in monolithic stationary phases or open tubular columns has given new opportunities for the development of miniaturized LC. The fabrication of monolithic structures inside microchannels has been demonstrated and is becoming a promising approach. However, monolithic stationary phases in microchannels require reproducible construction of uniform monoliths with low back pressure, which have not been adequately reduced to practice.

The use of capillaries for open tubular liquid chromatography (OTLC) was first proposed by Jorgenson et al. A key advantage of OTLC is lower back pressure than packed or monolithic columns, leading to faster analysis times. The main disadvantages of OTLC relative to packed column LC are slower mass transfer into the stationary phase and reduced sample capacity due to lower stationary phase volume. However, micromachined systems can have small channel cross sections, which increases the mass transfer to the stationary phase. Theoretical work on OTLC has shown that band dispersion is lowest for microchannels with high aspect ratios. Jacobson et al. reported the use of high aspect ratio microchannels to perform open channel electrochromatography. Later, the same group determined that 5 μm channel depths were a good compromise between efficiency and ease of operation. (See S. C. Jacobson, R. Hergenroder, L. B. Koutny and J. M. Ramsey, Anal. Chem., 1994, 66, 2369; and J. P. Kutter, S. C. Jacobson, N. Matsubara and J. M. Ramsey, Anal. Chem., 1998, 70, 3291.) However, these devices were not tested for pressure-driven separations.

In recent years, great interest has arisen in developing electrochemical systems for microchip pumping, resulting in devices for valve actuation and dosing systems for applications in biology and medicine. We have shown that the pressure caused by the build-up of electrolysis gases in an enclosed chamber can pump liquids in fluidic microchannels. (See J. W. Munyan, H. V. Fuentes, M. Draper, R. T. Kelly and A. T. Woolley, Lab Chip, 2003, 3, 217.) Our electrochemical micropumps are integrated easily with microfluidics and can pump liquids with rates as high as ˜10 μL/min. More recently, electrochemical actuation was demonstrated for sample delivery and solvent gradient generation in electrospray ionization mass spectrometry analysis. Only one report has appeared on the use of electrolysis-based micropumps in LC. While this initial work demonstrated feasibility, the separation time achieved was approximately one hour, which is similar to the separation times in conventional LC. Moreover, the electrolysis voltage was applied directly to the sample and eluent. (See J. Xie, Y. Miao, J. Shih, Y.-C. Tai and T. D. Lee, Anal. Chem., 2005, 77, 6947.)

SUMMARY OF THE INVENTION

A need exists for an apparatus, system, and method that integrates an electrochemical pump and a chromatography microcolumn in a single chip. Beneficially, such an apparatus, system, and method would provide short retention times and relatively high resolutions.

The present invention has been developed in response to the present state of the art, and in particular, in response to the problems and needs in the art that have not yet been fully solved by currently available chromatography systems including microcolumns and existing pumps for microfluidic systems. Accordingly, embodiments of the present invention have been developed to provide an apparatus, system, and method for electrochemical pump-based chromatography separations in microfabricated devices that overcome many or all of the shortcomings in the art.

In a simple form a microfluidic device in accordance with one embodiment of the invention includes a constant volume electrochemical pump formed in at least one layer of the microfluidic device. The microfluidic device includes a fluid reservoir coupled with the electrochemical pump for receiving pressure from the electrochemical pump during actuation. The microfluidic device also has a chromatographic microcolumn fluidically coupled to the fluid reservoir for receiving a fluid from the reservoir during actuation.

In another embodiment, the electrochemical pump may be a first injection pump and the fluid reservoir may be a first sample reservoir. In this case, the microfluidic device may further include at least one additional constant volume electrochemical pump formed in at least one layer of the device for pumping eluent. The microfluidic device may also include at least one additional reservoir fluidly coupled with the at least one additional electrochemical reservoir for receiving pressure from the additional electrochemical pump. The additional reservoir is also fluidically coupled to the chromatographic column.

In another embodiment, the microfluidic device may be formed of a plurality of layers. Electrodes may be disposed on or in at least one of the layers of the microfluidic device. In this embodiment, the electrodes form part of the electrochemical pump.

In one embodiment, the layers include a top layer having port holes therethrough in alignment with the electrochemical pump(s) and fluid reservoir(s). The port holes may be configured to receive an electrolyte and at least one of a sample and an eluent therethrough. The microfluidic device may further include a waste reservoir in the at least one of the layers. In any case, the waste reservoir is fluidically coupled to the chromatographic microcolumn for receiving at least a portion of the fluid after it passes through the microcolumn.

In another emobodiment, at least one layer is an intermediate layer and the microfluidic device further includes a top layer having port holes therethrough in alignment with the pump(s) and reservoir(s). The layers may further include a bottom layer. Thus, the intermediate layer may have the top layer and the bottom layer bonded thereto to form a monolithic pressure-driven liquid chromatography device. In still another embodiment, the microfluidic device may include a plurality of intermediate layers such that the pump(s) and reservoir(s) are formed by the plurality of intermediate layers.

In another simple form, a microfluidic system in accordance with embodiments of the invention includes a microcolumn in a chip. The microfluidic system also includes an electrochemical pumping mechanism in the chip. The electrochemical pumping mechanism includes at least one electrochemical pump fluidically coupled to a microcolumn. The electrochemical pumping mechanism supplies a sample under pressure to the column for chromatographic separation of the sample in the column. In one embodiment, electrochemical pumping mechanism includes at least one injection pump fluidly coupled to at least one sample reservoir and the microcolumn and at least one eluent pump fluidly coupled to at least one eluent reservoir and the microcolumn.

In another embodiment, each of the injection pump and the eluent pump includes a pair of electrodes configured to be in fluid communication with an electrolyte. In this embodiment, the couplings between the pumps and the reservoirs are configured such that when an electrolyte is present in each of the pumps, the electrolyte is fluidly connected to the sample and the eluent, respectively. With the configuration of this embodiment, gases generated by electrolysis apply pressure on at least one of the sample and the eluent via micro fabricated channels to effect pumping of the sample and/or the eluent in the reservoirs.

In another embodiment, at least one voltage source is connected to the electrodes in the injection pump and the eluent pump. The voltage source may be configured to control a voltage applied to respective pairs of electrodes. In one embodiment, a feedback loop and a current control device may be connected to the electrochemical pumping mechanism to automatically control a current supplied to the electrochemical pumping mechanism. In another embodiment, a feedback loop and a pressure monitoring device may be connected to the electrochemical pumping mechanism to automatically control pressure exerted by the electrochemical pumping mechanism.

In another simple form, a method for achieving fast liquid chromatography in a microdevice in accordance with embodiments of the present invention includes pumping at least one of a sample and an eluent by an electrochemical pump. The method also includes moving the sample through a microcolumn to a detection point in less than one minute. In one embodiment, moving the sample includes moving the sample through the microcolumn to the detection point in less than 40 seconds such that a presence of a material in the sample can be detected for an elution time of less than 40 seconds.

In one embodiment pumping includes applying an electrolysis voltage of 50 volts or less to an electrolyte in the electrochemical pump. In another embodiment, moving the sample through the microcolumn includes separating at least one material from another material along the microcolumn. The method may further include detecting the presence of at least one material.

In still another simple form, a method for achieving fast liquid chromatography in a microdevice in accordance with embodiments of the present invention includes balancing an injection of a sample with an injection of an eluent. Balancing the injections of sample and eluent includes controlling pressure of at least one of the sample and the eluent such that a predetermined amount of sample is injected in a microcolumn of a microdevice.

In one embodiment, the method includes selectively applying a voltage in at least one electrochemical pump fluidly coupled to at least one of the sample and the eluent. Selectively applying the voltage in the electrochemical pump may include applying a predetermined voltage to an electrolyte for a predetermined time. Alternatively, or additionally, selectively applying the voltage includes may include applying a first predetermined voltage to a first electrolyte in at least one electrochemical sample pump in fluid communication with the sample, and applying a second predetermined voltage to a second electrolyte in at least one additional electrochemical eluent pump in fluid communication with the eluent.

In another embodiment, the method may include monitoring a current supplied to the electrolyte, providing feedback to a controller, and automatically controlling the current supplied to the electrolyte. Alternatively, the method may include monitoring the pressure generated by the electrolyte, providing feedback to a controller, and automatically controlling the pressure generated by the electrolyte by changing the voltage, current, or time. Reference throughout this specification to features, advantages, or similar language does not imply that all of the features and advantages that may be realized with the present invention should be or are in any single embodiment of the invention. Rather, language referring to the features and advantages is understood to mean that a specific feature, advantage, or characteristic described in connection with an embodiment is included in at least one embodiment of the present invention. Thus, discussion of the features and advantages, and similar language, throughout this specification may, but do not necessarily, refer to the same embodiment.

Furthermore, the described features, advantages, and characteristics of the invention may be combined in any manner in one or more embodiments. The invention may be practiced without one or more of the specific features or advantages of a particular embodiment. In other instances, additional features and advantages may be recognized in certain embodiments that may not be present in all embodiments of the invention.

These features and advantages of the present invention will become more fully apparent from the following description and appended claims, or may be learned by the practice of the invention as set forth hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

In order that the advantages of the invention will be readily understood, a more particular description of the invention briefly described above will be rendered by reference to specific embodiments that are illustrated in the appended drawings. The drawings depict only typical embodiments of the invention and are not therefore to be considered to be limiting of its scope. The invention will be described and explained with additional detail with reference to the accompanying drawings, in which:

FIG. 1A is a schematic top plan view of a bottom, intermediate, and top layer of a chip showing patterns for etching, drilling, and metallization with a diagrammatic connection to a controller;

FIG. 1B is an exploded perspective view of the chip showing the layers of FIG. 1A in alignment for assembly

FIG. 1C is a perspective view of the chip of FIGS. 1A and 1B with the layers in their stacked and bonded condition after assembly;

FIG. 1D is a partial sectional view taken along line 1D-1D in FIG. 1C, showing an enclosed microchannel forming a microcolumn between layers in their assembled state;

FIG. 2 is a schematic representation in four progressive panels A-D of a pressure-balanced injection approach in which: (A) the microchannels are initially filled with eluent, then electrochemical pumps are charged with electrolyte; (B) the microcolumn is equilibrated by turning the eluent pump on while the sample pump remains off, (C) a sample is injected by pumping a portion of sample to the microchannel intersection with the sample pump on and the eluent pump off; (D) the sample is separated by turning the sample pump off and the eluent pump on to carry the portion of sample from the intersection through the microcolumn;

FIG. 3 is a graph showing results of chromatographic separation and detection for several repetitive runs using the microfluidic device and method of FIGS. 1A-2 to analyze a sample bearing a single analyte;

FIG. 4 is a graph showing results of chromatographic separation and detection of three replicated runs using the microfluidic device and method of FIGS. 1A-2 to analyze a sample bearing three amino acid analytes; and

FIG. 5 is a block diagram illustrating a method in accordance with an embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Reference throughout this specification to “one embodiment,” “an embodiment,” or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.

Furthermore, the described features, structures, characteristics, reagents, reaction times, temperatures, or atmospheric conditions of the invention may be combined in any suitable manner in one or more embodiments. In the following description, numerous specific details are provided, such as examples of programming, software modules, user selections, network transactions, database queries, database structures, hardware modules, hardware circuits, hardware chips, etc., to provide a thorough understanding of embodiments of the invention. One skilled in the relevant art will recognize, however, that the invention may be practiced without one or more of the specific details, or with other methods, components, materials, conditions and so forth. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the invention.

Rapid microchip LC analysis with integrated electrolysis-based pumping is achievable with microfabricated devices and a pressure balanced approach, as described herein. A combination microfluidic device may be integrated in a monolithic microfluidic chip. The pressure-balanced sample injection approach allows introduction of pL-range sample volumes without valves or other components that are difficult to integrate in microdevices. This approach also eliminates dead volume between the injection and separation modes. The on-chip LC separation of amino acids in accordance with embodiments of the invention have elution times of <40 s and good efficiency (3350 theoretical plates).

Many features of electrolysis, including simple instrumentation, rapid response time, low power consumption, and limited heat generation make it an attractive candidate for on-chip LC pumping. Moreover, a constant volume, electrolysis-based actuator can generate high pressures up to a maximum pressure of ˜200 MPa. In one embodiment of the invention, a flow rate of 210 nL/min (7.1 mm/s) was achieved with an electrolysis potential of 15 V. Furthermore, the ability of the micropumps to operate against elevated back pressure was tested. With a back pressure of 100 psi, a flow rate of 86 nL/min was still achieved. Embodiments of the present invention are capable of achieving flow rates in a range from 0.2 nL/min to 500 μL/min. Alternatively, the flow rates may be in a range from 1 nL/min to 50 μL/min. Further alternatively, the flow rates may be in a range from 5 nL/min to 5 μL/min. These ranges and any flow rate value are achievable with voltages in a range from 2 V to 100 V. Alternatively, the voltages may be in a range from 5 V to 25 V or in a range from 10 V to 20 B. In any case, embodiments of the present invention facilitate the miniaturization of pressure-driven separations or other pumping applications in which flow rates of nL/min-μL/min are required and in which these flow rates must be delivered in a simple and compact format

Configuration, fabrication, and characterization of electrically actuated micropumps for pressure-driven LC in microchips are set forth herein. Micropumps may be formed in glass and integrated with microfabricated channels in the same substrate by etching or other material removal mechanisms. Any of a variety of etching methods and etchants may also be used. Substrates may include any of a variety of materials. Microfeatures for devices may be placed in alternative or additional substrate materials including silicon, glass, quartz, plastics, semiconductors, insulators, metals, and combinations thereof. A pressure-balanced injection approach may be implemented by controlling the electrolysis time and voltage applied to the eluent and sample micropumps. There are parameters that affect the reproducibility of the system. In this regard, the column coating, amount of sample injected, and eluent composition may be adjusted in order to establish the effects from respective values for these parameters. The function of devices configured in accordance with embodiments of the present invention may be tested with samples containing fluorescently labeled amino acids. Thus, electrolysis-based injection and LC separation of fluorescently labeled amino acids can be evaluated.

FIG. 1A is a schematic top plan view of a bottom, intermediate, and top layer of a chip 13 showing patterns of etching, drilling, and metalization with a diagrammatic connection to a controller 14. FIG. 1A shows the chip 13 in a pre-assembled condition with a bottom layer 15, an intermediate layer 18, and a cover or top layer 21. The bottom layer 15 may include an injection microchannel 23 for sample injection, an eluent transfer microchannel 24 for loading eluent, and a separation microchannel 25 for separation. The bottom layer may also include a pair of electrodes 27, 28 for sample loading and a pair of electrodes 29, 30 for eluent pumping. Both sets of electrode pairs 27, 28 and 29, 30 are positioned for alignment with structure on the intermediate layer forming a sample pump and an eluent pump (to be described below).

The intermediate layer 18 may be one of a plurality of similar intermediate layers. The intermediate layer(s) 18 include reservoir through-holes 33, 36 that form the reservoirs for sample and eluent. The intermediate layers also include pump through-holes 39, 42 that form receptacles of an electrolyte solution. A pressure transfer tube 45 on top of an upper one of the intermediate layers 18 connects the sample pump through-hole 39 with the sample reservoir 33 and another pressure transfer tube 48 connects the eluent pump through-hole 42 with the eluent reservoir 36. The intermediate layer(s) also have a waste reservoir through-hole 51 for receiving fluid after it passes from at least one of the sample and eluent reservoirs 33, 36, through at least one of the injection microchannel 23 and eluent transfer microchannel 24, and through the separation microchannel 25.

The top layer 21 may include access openings 53, 54, 55, 56, 57 positioned to be in alignment with the sample reservoir 33, sample through-hole 39, eluent reservoir 36, eluent pump through-hole 42, and waste reservoir through-hole 51, respectively, to facilitate the introduction of sample, eluent and electrolyte solution into their reservoirs or receptacles. In one embodiment, the sample, eluent, and waste reservoirs are formed as three 2.5 mm diameter through-holes. In this embodiment, the sample pump and eluent pump through-holes are two 1.0 cm diameter reservoirs. The access openings 53, 54, 55, 56, 57 are formed as five 1.5 mm diameter holes. Through-holes of other sizes and configurations may be utilized in addition to or in place of those shown and described herein.

The layers 15, 18, and 21 may be cleaned, etched with isotropic or other types of etching, metallized in the electrode areas, and drilled to form the various features described above. The various features may be provided by other methods including molding, machining, and/or micromachining, for example.

The injection microchannels 23, 24 may have lengths of 2 cm or less. The separation microchannel 25 may have a length of 20 cm or less. In one embodiment, the injection microchannels are approximately 1 cm long and the separation microchannel is approximately 3 cm long. In this embodiment, the microchannels 23, 24, 25 have a width of approximately 100 μm and a depth of approximately 5 μm. The injection and separation microchannels 23, 24, 25 may have a width in a range from 5 μm to 300 μm, and a depth in a range from 2 μm to 10 μm. Alternatively, the width may be in a range from 10 μm to 150 μm, and the depth may be in a range from 3 μm to 8 μm. Further alternatively, the width may be in a range from 20 μm to 100 μm, and the depth may be in a range from 4 μm to 6 μm. The width and depth may be of any value within these ranges. In one embodiment, the pressure transfer tubes 45, 48 have a length of approximately 1 cm, although the pressure transfer tubes may have greater or lesser lengths. The pressure transfer tubes 45, 48 may have a width in a range from 20 μm to 500 μm, and a depth in a range from 10 μm to 200 μm. In one embodiment, the width of the pressure transfer tubes is approximately 200 μm.

The electrodes 27, 28, 29, 30 may be thermally or otherwise deposited on the bottom layer 15, and may include gold, platinum, conductive epoxy, and/or other electrically conductive materials. In one embodiment, the electrodes are formed as four 1.5×9 mm rectangular thin layers. Electrical connections between a controller 14 and the electrodes 27, 28, 29, 30 may include platinum or other conductive wires 59 and conductive epoxy. In another embodiment, platinum wires are inserted through holes formed in the bottom layer 15 and are bonded in positions with exposed wire extending on a top surface of the bottom layer at locations corresponding to the pump through-holes 39,42. The wires are sealed and held in position by an epoxy material. In other embodiments the electrodes may be formed as interdigitated electrodes, in a three-electrode format (including reference electrode), to control the current or voltage. In a further alternative or additional variation, the electrodes may include one or more electrodes comprised of a material such as palladium that can absorb one of the gases produced in the electrolysis. In some embodiments, the electrodes are provided with greater surface area to improve the stability of the current supplied to the electrodes 27, 28, 29, 30. Additionally or alternatively, the electrodes could be connected to feedback loops 60, 61 and a controller 14 that includes a current controller 63 for automatically controlling the current supplied to the electrodes 27, 28, 29, 30. Additionally or alternatively, the electrodes could be connected to feedback loops 60, 61 and a controller 64 that includes a pressure monitor 65 automatically controlling the pressure in reservoirs 33 and 36.

FIG. 1B is an exploded perspective view of the chip 13 showing the layers 15, 18, 21 of FIG. 1A in alignment for assembly in a stacked, bonded configuration. The layers 15, 18, 21 may be trimmed, cleaned, and manually aligned for bonding. Volumes of the reservoirs and pumps may be increased by including additional intermediate layers 18, as indicated above. Once properly aligned the layers 15, 18, 21 are clamped and heated in a furnace in order to bond the layers together.

FIG. 1C is a perspective view of the chip 13 of FIGS. 1A and 1B with the layers 15, 18, 21 in their stacked and bonded condition after assembly. FIG. 1C shows a perspective view of the resultant chip 13 and a corresponding monolithic microfluidic device. The monolithic microfluidic device is actually made up of a plurality of microfluidic devices. In the illustrated embodiment the plurality of microfluidic devices include an injection or sample micropump 66, an eluent micropump 68, and a microchannel or microcolumn 71. It is to be understood that in other embodiments additional pumps and/or reservoirs that are not shown in FIG. 1C may be included without limitation. FIGS. 1A-1C also show port openings or access openings 53, 54, 55, 56, and 57 in alignment with each of the respective through-holes 33, 39, 36, 42, and 48 including the reservoir through-holes 33, 36, and 51 forming sample, eluent, and waste reservoirs 74, 75, and 76, respectively. The reservoirs 74, 75, 76 and the micropumps 66, 68 are closed off at lower ends thereof by appropriate portions of the bottom layer 15. Thus, the electrodes 27, 28 underlie the sample pump through-hole 39 and the electrodes 29, 30 underlie the eluent pump through-hole 42. These electrodes 27, 28, 29, 30 form part of the sample and eluent micropumps 66, 68, respectively. Also, the injection microchannel 23 and eluent transfer microchannel 24 extend from the separation microchannel 25 to the lower ends of the reservoir through-holes 33, 36, and the transfer tubes 45, 48 extend from top ends of the reservoir through-holes 33, 36 to the pump through-holes 39, 42 when the layers 15, 18, 21 are in the assembled state.

The resultant chip 13 may have ports 78 or plugs 79, examples of which are shown inserted in the access openings 53, 54, 55, 56, and 57 of the chip 13. The plugs 79 can be disposed therein to seal an interior and create a constant volume micropump device, for example. The ports 78 may be in the form of Nanoport reservoirs (Upchurch Scientific, Oak Harbor, Wash.) disposed in the access openings to facilitate introduction of electrolyte, sample, and eluent fluids into the device. The plugs 79 may be in the form of sealing nuts (Upchurch). It is to be understood that the access openings are converted to “ports” at appropriate times during use in order to introduce sample, eluent or electrolyte. Once appropriately loaded, the ports 78 can be removed and the access openings capped with sealing nuts to operate as plugs 79.

FIG. 1D is a partial sectional view taken along line 1D-1D in FIG. 1C, showing the microchannel 25 enclosed by a lower surface of the intermediate layer 18 and forming a chromatography microcolumn 71 between layers 15, 18 in their assembled state. The separation microchannel 25 is converted into the chromatography microcolumn 71 by applying a coating 80 on its walls. The walls of the chromatography microcolumn 71 thus comprise the coated walls of the separation microchannel 25 and a portion of an underside of a lowermost layer of the intermediate layers 18 that covers and encloses the separation microchannel 25. The result is that the microcolumn 71 forms a microcapillary, a partial sectional view of which is shown in FIG. 1D.

As discussed above, the microchannels 23, 24, 25 may be formed by an etching process that determines a width and a depth of the channels. For example, with a mask that leaves an unprotected portion having a width of approximately 100 μm, wet chemical isotropic etching can be used to produce microchannel widths of approximately 104-112 μm and microchannel depths of approximately 2-6 μm. As shown in FIG. 1D, the bond between the intermediate layer 18 and the bottom layer 15 may be so complete that an interface line becomes invisible. Thus, the layers become one-piece. In the cross sectional view of FIG. 1D, a relatively large width 82 to depth 83 aspect ratio is depicted. However, the width to depth aspect ratio may be in a range from 2 to 100. Alternatively, the width to depth aspect ratio may be in a range from 5 to 50. Still further alternatively, the width to depth aspect ratio may be in a range from 10 to 25.

The walls forming the microcolumn 71 may be treated chemically or otherwise to receive the coating 80. In one embodiment, the microcolumn is chemically treated to create a coating of octadecylsilane in accordance with the method of Kutter et al. (See J. P. Kutter, S. C. Jacobson, N. Matsubara and J. M. Ramsey, Anal. Chem., 1998, 70, 3291.) This method can be adapted to formation of columns in accordance with embodiments of the present invention. For example, after passing through a 0.45 μm filter, the coating solution may be aspirated via the waste reservoir 51 through the separation microchannel 25 for 12 hours at room temperature. Vacuum (as opposed to pressure) may be applied for surface derivatization to inhibit stationary phase deposition and analyte retention in the injection microchannel 23.

The schematic flow diagrams that follow are generally set forth as logical flow diagrams. As such, the depicted order and labeled operations are indicative of one embodiment of the presented method. Other operations and methods may be conceived that are equivalent in function, logic, or effect to one or more operations, or portions thereof, of the illustrated method. Additionally, the format and symbols employed are provided to explain the logical operations of the method and are understood not to limit the scope of the method. Although various arrow types and line types may be employed in the flow diagrams, they are understood not to limit the scope of the corresponding method. Indeed, some arrows or other connectors may be used to indicate only the logical flow of the method. For example, an arrow or boundary line between blocks or panels may indicate a waiting or monitoring period of unspecified duration between enumerated operations of the depicted method. Additionally, the order in which a particular method occurs may or may not strictly adhere to the order of the corresponding operations shown.

FIG. 2 is a schematic representation in four progressive panels A-D of a pressure-balanced injection approach in which: (A) the microchannels are initially filled with eluent, then electrochemical pumps are charged with electrolyte; (B) microfluidic channels are equilibrated by turning the eluent pump on while the sample pump remains off; (C) a sample is injected by pumping a portion of sample to the microchannel intersection with the sample pump on and the eluent pump off; and (D) the sample is separated by turning the sample pump off and the eluent pump on to carry the portion of sample from the intersection through the microcolumn. As such, FIG. 2 is a diagrammatic view of operations in a method of performing pressure-balanced injection. This method is generally implemented by independently controlling the electrolysis time and voltage applied in the electrochemical micropumps 66, 68.

By way of example for sample injection and separation, panel A of FIG. 2 shows how the microchannels may be filled by pipetting of eluent 89 via the ports into the eluent reservoir 75 and sample reservoir 74. Vacuum may be applied to the waste reservoir 76 to draw the eluent through the microchannels, as indicated by vacuum pump 92. Then, an electrolyte solution 95 may be loaded via the ports 78 into the sample pump 66 and the eluent pump 68. After loading the electrolyte 95, the sample and eluent pump access openings 54, 56 may be capped by the plugs 79, which may take the form of sealing nuts.

Panel B in FIG. 2 depicts how the eluent 89 in the sample reservoir 74 may be removed and replaced with a sample 98. Both the sample reservoir 33 and the eluent reservoir 36 may then also be closed and sealed by plugs 79. In order to equilibrate the device in accordance with this example, a low voltage (eg., ˜20 V) may be applied to the eluent pump 68 for a short period (eg., ˜5 s) until the injection microchannel 23, the eluent transfer microchannel 24, and the separation microchannel 25 are bubble-free and filled with eluent 89, as shown in panel B of FIG. 2.

Then, for injection as shown in panel C of FIG. 2, a low voltage (eg., ˜20 V) may be applied to the sample pump 66 while the voltage applied to the eluent pump 68 is turned off. After an empirically determined time, (eg., ˜5-8 s), a plug of sample 98 is transferred to an intersection 101 of the microchannels 23, 24, 25.

Then the sample pump 66 may be turned off, as shown in panel D of FIG. 2, and a low voltage (eg., ˜25 V) may be applied to the eluent pump 68 to move the injected plug of sample 98 through the separation column, as indicated by arrow 104. As shown at 107 in panel D, the plug of sample 98 separates due to different amounts of dwell time spent by different materials within the sample 98 in the stationary phase versus in the mobile phase in the microcolumn 71.

Embodiments of the method may include labeling a material in the sample with fluorescein-5-isothiocyanate (FITC) or any of a variety of other detectable labels. The method may also include detecting the labeled material at a predetermined location along the column 71, as indicated by arrow 108 in FIG. 2. With FITC labels for example, laser-induced fluorescence detection may be conducted in the separation microchannel or microcolumn 71 at a distance (eg., ˜2.5-cm) from the injection intersection. This method of detection is described by Kelly and Woolley. (See R. T. Kelly and A. T. Woolley, Anal. Chem., 2003, 75, 1941, which is incorporated herein by reference.) Alternatively, electrochemical, conductance, absorbance, or other detection modalities can be implemented.

In one embodiment of the method, an injection time for a sample plug to move from the sample reservoir 74 to the microchannel intersection 101 may be in a range from 5 s to 8 s. The amount of the sample 95 loaded into the separation microchannel 25 can be controlled by adjusting a pumping time of the sample pump 66. Thus, adjusting the pumping time defines the length of the injected sample plug. For example, in one embodiment, an injection time of 6 s injected a plug having a length, which when measured at the full width at half maximum was estimated to be 90-100 μm. This corresponds to an injection volume of ˜50 pL.

FIG. 3 is a graph 109 showing results of chromatographic separation and detection of several repetitive runs using the microfluidic device and method of FIGS. 1A-2 to analyze a sample bearing a single analyte. The runs depicted in FIG. 3 are the results of a test of the reproducibility of the pressure balanced injection described above. As shown, relatively narrow, well-defined peaks are shown for repetitive injections of a sample bearing a single analyte bearing an FITC label and 70:30 acetonitrile/50 mM acetate buffer (pH 5.45) as the eluent. As can be seen from the graph 109, the retention times in this example are approximately 30 s. Thus, it is noted that at least some embodiments of the pumping system are best suited for relatively fast (<1 min) microchip LC analysis.

FIG. 4 is a graph 110 showing results of chromatographic separation and detection of three replicated runs using the microfluidic device and method of FIGS. 1A-2 to analyze a sample bearing three amino acid analytes. The graph 110 of FIG. 4 shows the results of a test of the performance of the LC microdevices in accordance with embodiments of the present invention. In this case, separation of three FITC-labeled amino acids is shown. The graph 110 of FIG. 4 presents chromatograms of three replicate electrolysis-based pressure-driven LC microchip separations of aspartic acid, glycine and phenylalanine having elution times of about 35 s or less. The peak identities were determined by comparing them to elution times of individually injected amino acids, and are consistent with the polarities of these analytes.

It is noted that embodiments of the method include achieving a reproducibility of the retention time for aspartic acid, and reproducibility for other analytes is likely achievable by eliminating device induced deviations. The relative standard deviation (RSD) for the retention time of the first peak (corresponding to aspartic acid) was 2.2%. An efficiency (N) of 3350 theoretical plates was obtained for this aspartic acid peak, which corresponds to a plate height (H) of 7.5 μm for the 2.5 cm separation channel used in this test. This value improves over prior plate heights (12-50 μm) reported for microchip OTLC without integrated pumping. (See P. G. Vahey, S. H. Park, B. J. Marquardt, Y. Xia, L. W. Burgess and R. E. Synovec, Talanta, 2000, 51, 1205.) An advantage of embodiments of the present invention is that they allow the injection of pL-range samples with no dead volume between the injector and column, which improves efficiency in microchip LC.

The peaks shown in the graph 110 of FIG. 4 have either baseline or nearly baseline separation. The resolution between aspartic acid and glycine is approximately 1.2 without gradient elution or longer columns. It is noted that improved resolution could be achieved by enabling gradient elution. This can be done by incorporating another elution pump to deliver a second eluent in the system. On the other hand, the separations achieved by embodiments of the present invention are approximately 20 fold faster than those achieved by conventional LC. Furthermore, the separation efficiency in embodiments of the present invention can be improved by incorporating double-etched profiles near the column sidewalls to reduce band dispersion.

FIG. 5 is a block diagram illustrating a method 111 for performing micro level liquid chromatography in accordance with an embodiment of the present invention. One of the operations in the method 111 is pumping by an electrochemical pump, as indicated at 113. This operation may include applying an electrolysis voltage of less than 50 V, as indicated at 116. Alternatively, the electrolysis voltage applied may be less than 25 V. Further alternatively, the electrolysis voltage applied may be less than 10 V. The method 111 also includes moving a sample through a column, which in this case is a microcolumn, as indicated at 119. Moving the sample through the column may include separating one material from another, as indicated at 120. In one embodiment, the method includes pumping by an electrochemical pump and moving the sample through the column in an integral or monolithic chip.

The method of FIG. 5 may also include balancing sample and eluent injection, as indicated at 123. This balancing may include selectively applying one or more voltages to one or more electrolytes in electrolyte-based pumps, as indicated at 127. For example, one or more sample pumps may be selectively turned on or off, as indicated at 130. Alternatively or additionally, one or more eluent pumps may be turned on or off, as indicated at 133. Selectively applying voltage(s) also includes selectively applying the voltages for predetermined times or times determined in real time.

In one embodiment, the method includes monitoring and/or controlling current supplied to one or more electrolytes in electrolyte-based pumps, as indicated at 136. Alternatively, the currents may be monitored and/or controlled in other electrochemical pumps. The operation of monitoring and controlling the currents may be implemented during any of the other operations. Relatedly, the method may include sensing a pressure in one or more of the reservoirs and providing feedback to a pressure controller. Based on the pressure detected in the reservoir(s), the current and resulting pressure can be adjusted. The method may also include detecting a material in the sample, as indicated at 139.

The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments are to be considered in all respects only as illustrative and not restrictive. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope. 

1. A microfluidic device comprising: a constant volume electrochemical pump formed in at least one layer of a microfluidic device; a fluid reservoir coupled with the electrochemical pump for receiving pressure from the electrochemical pump during actuation; and a chromatographic microcolumn fluidically coupled to the fluid reservoir for receiving a fluid from the reservoir during actuation.
 2. The microfluidic device of claim 1, wherein the chromatographic column has an aspect ratio of width to height in a range from 2 to
 100. 3. The microfluidic device of claim 1, wherein the electrochemical pump is a first injection pump and the fluid reservoir is a first sample reservoir, the microfluidic device further comprising: at least one additional constant volume electrochemical pump formed in the at least one layer for pumping eluent; and at least one additional reservoir fluidly coupled with the at least one additional electrochemical reservoir for receiving pressure from the additional electrochemical pump, the additional reservoir fluidically coupled to the chromatographic column.
 4. The microfluidic device of claim 1, further comprising: another layer forming the microfluidic device; electrodes disposed on at least one of the layers of the microfluidic device, the electrodes forming part of the electrochemical pump.
 5. The microfluidic device of claim 1, further comprising a top layer having port holes therethrough in alignment with the electrochemical pump and fluid reservoir, the port holes configured to receive an electrolyte and at least one of a sample and an eluent therethrough.
 6. The microfluidic device of claim 1, further comprising a waste reservoir in the at least one layer, the waste reservoir fluidically coupled to the chromatographic microcolumn for receiving at least a portion of the fluid after it passes through the microcolumn.
 7. The microfluidic device of claim 1, wherein the at least one layer is an intermediate layer, the microfluidic device further comprising: a top layer having port holes therethrough in alignment with the pump and reservoirs, and a bottom layer; wherein the intermediate layer has the top layer and the bottom layer bonded thereto to form a monolithic pressure-driven liquid chromatography device.
 8. The microfluidic device of claim 7, further comprising a plurality of intermediate layers such that the pump and reservoirs are formed by the plurality of intermediate layers.
 9. A microfluidic system, comprising: a microcolumn in a chip; and an electrochemical pumping mechanism in the chip; the electrochemical pumping mechanism having at least one electrochemical pump fluidically coupled to a microcolumn; wherein the electrochemical pumping mechanism supplies a sample under pressure to the column for chromatographic separation of the sample in the column.
 10. The microfluidic system of claim 9, wherein the electrochemical pumping mechanism comprises: at least one injection pump fluidicly coupled to at least one sample reservoir and the microcolumn; and at least one eluent pump fluidicly coupled to at least one eluent reservoir and the microcolumn.
 11. The microfluidic system of claim 10, wherein: each of the injection pump and the eluent pump comprises a pair of electrodes configured to be in fluid communication with an electrolyte; and the couplings between the pumps and the reservoirs are configured such that when an electrolyte is present in each of the pumps, the electrolyte is fluidly connected to the sample and the eluent, respectively; whereby gases generated by electrolysis apply pressure on at least one of the sample and the eluent via microfabricated channels to effect pumping of the sample and/or the eluent in the reservoirs.
 12. The microfluidic system of claim 11, further comprising at least one voltage source connected to the electrodes in the injection pump and the eluent pump, the voltage source configured to control a voltage applied to respective pairs of electrodes.
 13. The microfluidic system of claim 9, further comprising a feedback loop and a current control device connected to the electrochemical pumping mechanism to automatically control a current supplied to the electrochemical pumping mechanism.
 14. The microfluidic system of claim 9, further comprising a feedback loop and a pressure monitoring device connected to the electrochemical pumping mechanism to automatically control pressure exerted by the electrochemical pumping mechanism.
 15. A method for achieving fast liquid chromatography in a microdevice, the method comprising: pumping at least one of a sample and an eluent by an electrochemical pump; and moving the sample through a microcolumn to a detection point in less than one minute.
 16. The method of claim 15, wherein moving the sample comprises moving the sample through the microcolumn to the detection point in less than 40 seconds such that a presence of a material in the sample can be detected for an elution time of less than 40 seconds.
 17. The method of claim 15, wherein pumping comprises applying an electrolysis voltage of 50 volts or less to an electrolyte in the electrochemical pump.
 18. The method of claim 15, wherein moving the sample through the microcolumn comprises separating at least one material from another material along the microcolumn, the method further comprising detecting the presence the at least one material.
 19. A method for achieving fast liquid chromatography in a microdevice, the method comprising balancing an injection of a sample with an injection of an eluent, wherein balancing the injections of sample and eluent comprises controlling pressure of at least one of the sample and the eluent such that a predetermined amount of sample is injected in a microcolumn of a microdevice.
 20. The method of claim 19, further comprising selectively applying a voltage in at least one electrochemical pump fluidly coupled to at least one of the sample and the eluent.
 21. The method of claim 20, wherein selectively applying the voltage in the electrochemical pump comprises applying a predetermined voltage to an electrolyte for a predetermined time.
 22. The method of claim 21, further comprising monitoring a current supplied to the electrolyte, providing feedback to a controller, and automatically controlling the current supplied to the electrolyte.
 23. The method of claim 21, further comprising monitoring pressure generated by the electrolyte, providing feedback to a controller, and automatically controlling the pressure generated by the electrolyte by changing at least one of the voltage, current, or time.
 24. The method of claim 20, wherein selectively applying the voltage comprises: applying a first predetermined voltage to a first electrolyte in at least one electrochemical sample pump in fluid communication with the sample; and applying a second predetermined voltage to a second electrolyte in at least one additional electrochemical eluent pump in fluid communication with the eluent. 